Myeloperoxidase, a risk indicator for cardiovascular disease

ABSTRACT

Diagnostic tests for characterizing an individual&#39;s risk of developing or having a cardiovascular disease. In one embodiment the present diagnostic test comprises determining the level of myeloperoxidase (MPO) activity in a bodily sample obtained from the individual or test subject. In another embodiment, the diagnostic test comprises determining the level of MPO mass in a bodily sample obtained from the test subject. In another embodiment, the diagnostic test comprises determining the level of one or more select MPO-generated oxidation products in a bodily sample obtained from the test subject. The select MPO-generated oxidation products are dityrosine, nitrotyrosine, methionine sulphoxide or an MPO-generated lipid peroxidation products. Levels of MPO activity, MPO mass, or the select MPO-generated oxidation product in bodily samples from the test subject are then compared to a predetermined value that is derived from measurements of MPO activity, MPO mass, or the select MPO-generated oxidation product in comparable bodily samples obtained from the general population or a select population of human subjects. Such comparison characterizes the test subject&#39;s risk of developing CVD.

This application claims priority to U.S. Provisional Application60/259,340 filed Jan. 2, 2001 and U.S. Provisional Application60/283,432 filed May 12, 2001, both of which are incorporated herein intheir entirety.

The work described in this application was supported, at least in part,by Grant No. RO1 HL62526-01 from the National Institutes of Health. TheUnited States government has certain rights in this invention.

BACKGROUND

The present invention relates to the field of cardiovascular disease.More specifically, it relates to a diagnostic test which can be used todetermine whether an individual or test subject is at a lower risk orhigher risk of developing or having cardiovascular disease than otherindividuals in a given population of human subjects.

Cardiovascular disease (CVD) is the general term for heart and bloodvessel diseases, including atherosclerosis, coronary heart disease,cerebrovascular disease, and peripheral vascular disease. Cardiovasculardisorders are acute manifestations of CVD and include myocardialinfarction, stroke, angina pectoris, transient ischemic attacks, andcongestive heart failure. CVD accounts for one in every two deaths inthe United States and is the number one killer disease. Thus, preventionof cardiovascular disease is an area of major public health importance.

A low fat diet and exercise are recommended to prevent CVD. In addition,a number of drugs may be prescribed by medical professionals to thosepersons who are known to be at risk for developing CVD. These includelipid lowering agents which reduce blood levels of cholesterol andtrigylcerides. Medications to normalize blood pressure are used inhypertensive patients. Medications which prevent activation ofplatelets, such as aspirin, may also be prescribed for patients at riskfor developing CVD. More aggressive therapy, such as administration ofmultiple medications, may be used in those individuals who are at highrisk. Since CVD therapies may have adverse side effects, it is desirableto have diagnostic tests for identifying those individuals who are atrisk, particularly those individuals who are at high risk, of developingCVD.

Currently, several risk factors are used by members of the medicalprofession to assess an individual's risk of developing CVD and toidentify individuals at high risk. Major risk factors for cardiovasculardisease include hypertension, family history of premature CVD, smoking,high total cholesterol, low HDL cholesterol, and diabetes. The majorrisk factors for CVD are additive, and are typically used together byphysicians in a risk prediction algorithm to target those individualswho are most likely to benefit from treatment for CVD. These algorithmsachieve a high sensitivity and specificity for predicting 15% risk ofCVD within 10 years. However, the ability of the present algorithms topredict a higher probability of developing CVD is limited. Among thoseindividuals with none of the current risk factors, the 10-year risk fordeveloping CVD is still about 2%. In addition, a large number ofcardiovascular disorders occur in individuals with apparently low tomoderate risk profiles, as determined using currently known riskfactors. Thus, there is a need to expand the present cardiovascular riskalogrithm to identify a larger spectrum of individuals at risk for oraffected with CVD.

The mechanism of atherosclerosis is not well understood. Over the pastdecade a wealth of clinical, pathological, biochemical and genetic datasupport the notion that atherosclerosis is a chronic inflammatorydisorder. Acute phase reactants (e.g. C-reactive protein, complementproteins), sensitive but non-specific markers of inflammation, areenriched in fatty streaks and later stages of atherosclerotic lesions.In a recent prospective clinical trial, base-line plasma levels ofC-reactive protein independently predicted risk of first-time myocardialinfarction and stroke in apparently healthy individuals. U.S. Pat. No.6,040,147 describes methods which use C-reactive protein, cytokines, andcellular adhesion molecules to characterize an individual's risk ofdeveloping a cardiovascular disorder. Although useful, these markers maybe found in the blood of individuals with inflammation due to causesother than CVD, and thus, these markers are not highly specific.

Accordingly, the need still exits for additional diagnostic tests forcharacterizing an individuals risk of developing or of havingcardiovascular disease. Diagnostic tests which employ risk factors thatare independent of traditional CVD risk factors such as LDL levels areespecially desirable.

SUMMARY OF THE INVENTION

The present invention provides new diagnostic tests for characterizingan individual's risk of developing or having cardiovascular disease. Thepresent tests are especially useful for identifying those individualswho are in need of highly aggressive CVD therapies as well as thoseindividuals who require no therapies targeted at preventing CVD. Thepresent diagnostic tests are based on the discovery that patients withcoronary artery disease (CAD) have significantly greater levels ofleukocyte and blood myeloperoxidase (MPO) levels than patients withoutangiographically significant CAD. It has also been discovered thatleukocyte-MPO levels in CAD and non-CAD patients are independent of age,sex, diabetes, hypertension, smoking (ever or current), WBC count,LDL-C, trigylcerides, and Framingham Global Risk Score. Thus, thepresent diagnostic tests, which involve assessing levels of MPOactivity, MPO mass, or levels of select MPO-generated oxidation productsin a blood sample or derivative thereof from a test subject, provideadditive predictive value beyond that seen with clinical and diagnosticrisk factors currently employed by physicians.

In one aspect, the present diagnostic test comprises determining thelevel of MPO activity in a bodily sample obtained from the individual ortest subject. The bodily sample is blood or a derivative thereof,including but not limited to, leukocytes, neutrophils, monocytes, serum,or plasma. The level of MPO activity in the bodily sample from the testsubject is then compared to a predetermined value that is derived frommeasurements of MPO activity in comparable bodily samples obtained fromthe general population or a select population of human subjects. Suchcomparison characterizes the test subject's risk of developing CVD. Forexample, test subjects whose blood levels of MPO activity are higherthan the predetermined value are at greater risk of developing or havingCVD than individuals whose blood MPO activity levels are at or lowerthan the predetermined value. Moreover, the extent of the differencebetween the test subjects MPO activity levels and predetermined value isalso useful for characterizing the extent of the risk and thereby,determining which individuals would most greatly benefit from certaintherapies.

In another aspect, the diagnostic test comprises determining the levelof MPO mass in a bodily sample obtained from the test subject. Thebodily sample is blood or a derivative thereof, including but notlimited to, leukocytes, neutrophils, monocytes, serum, or plasma. Levelsof MPO mass in bodily samples from the test subject are then compared toa predetermined value that is derived from measurements of MPO mass incomparable bodily samples obtained from healthy controls. Suchcomparison characterizes the test subject's risk of developing CVD.

In another aspect, the diagnostic test comprises determining the levelof one or more select MPO-generated oxidation products in a bodilysample obtained from the test subject. The select MPO-generatedoxidation products are dityrosine, nitrotyrosine, methionine sulphoxide,and MPO-generated lipid peroxidation products. Preferred MPO lipidperoxidation products are hydroxy-eicosatetraenoic acids (HETEs);hydroxy-octadecadienoic acids (HODEs); F2Isoprostanes; the glutaric andnonanedioic monoesters of 2-lysoPC (G-PC and ND-PC, respectively); the9-hydroxy-10-dodecenedioic acid and 5-hydroxy-8-oxo-6-octenedioic acidesters of 2-lysoPC (HDdiA-PC and HOdiA-PC, respectively); the9-hydroxy-12-oxo-10-dodecenoic acid and 5-hydroxy-8-oxo-6-octenoic acidesters of 2-lysoPC (HODA-PC and HOOA-PC, respectively); the9-keto-12-oxo-10-dodecenoic acid and 5-keto-8-oxo-6-octenoic acid estersof 2-lysoPC (KODA-PC and KOOA-PC, respectively); the9-keto-10-dodecendioic acid and 5-keto-6-octendioic acid esters of2-lysoPC (KDdiA-PC and KOdiA-PC, respectively); the 5-oxovaleric acidand 9-oxononanoic acid esters of 2-lysoPC (OV-PC and ON-PC,respectively); 5-cholesten-5α, 6α-epoxy-3β-ol (cholesterol α-epoxide);5-cholesten-5β, 6β-epoxy-3β-ol (cholesterol β-epoxide);5-cholesten-3β,7β-diol (7-OH-cholesterol); 5-cholesten-3β, 25-diol(25-OH cholesterol); 5-cholesten-3β-ol-7β-hydroperoxide (7-OOHcholesterol); and cholestan-3β, 5α, 6β-triol (triol). The bodily sampleis blood, urine or a blood derivative, including but not limited to,leukocytes, neutrophils, monocytes, serum, or plasma. Levels of theselected MPO-generated oxidation products in bodily samples from thetest subject are then compared to a predetermined value that is derivedfrom measurements of the selected MPO-generated oxidation products incomparable bodily samples obtained from healthy controls. Suchcomparison characterizes the test subject's risk of developing CVD.

For those individuals who have already experienced an acute adversecardiovascular event such as a myocardial infarction or ischemic stroke,the present diagnostic tests are also useful for assessing suchindividual's risk of having a recurrent event. Thus, the presentinvention also provides a method for monitoring over time the status ofCVD in a subject. The method comprises determining the levels of one ormore of the present risk factors, including MPO activity, MPO mass,select MPO-generated oxidation products, and combinations thereof, in abodily sample taken from the subject at an initial time and in acorresponding bodily fluid taken from the subject at a subsequent time.An increase in the levels of the present risk factors from the bodilyfluid taken at the subsequent time as compared to the initial timeindicates that a subject's risk of having a future cardiovascularevent/disorder has increased. A decrease in the levels of the presentrisk factors from the bodily fluid taken at the subsequent time ascompared to the initial time indicates that that the subject's risk ofhaving a cardiovascular event has decreased.

In another aspect, the present invention provides a method forevaluating therapy in a subject suspected of having or havingcardiovascular disease. The method comprises determining the levels ofone or more of the present risk factors, including MPO activity, MPOmass, select MPO-generated oxidation products, and combinations thereof,in a bodily sample taken from the subject prior to therapy and acorresponding bodily fluid taken from the subject during or followingtherapy. A decrease in the level of the selected risk factor in thesample taken after or during therapy as compared to the level of theselected risk factor in the sample taken before therapy is indicative ofa positive effect of the therapy on cardiovascular disease in thetreated subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A kinetic model for myeloperoxidase.

FIG. 2. A schematic representation of certain myeloperoxidase generatedreactive intermediates and some MPO-generated oxidation products.

FIG. 3. The chemical structure of dityrosine and nitrotyrosine.

FIG. 4. Lipid Peroxidation in Plasma with Neutrophils from HealthySubjects and MPO Deficient Subjects. Neutrophils (1×10⁶/ml) isolatedfrom normal and MPO-deficient individuals were incubated at 37° C. inHBSS supplemented with DTPA (100 μM, pH 7.0) and fresh human plasma (50%v/v). Cells were activated by addition of phorbol myristate acetate(PMA, 200 nM) and incubated for 2 h (Complete System). The content of9-H(P)ODE and 9-H(P)ETE formed within endogenous plasma lipids were thendetermined by LC/ESI/MS/MS. Where indicated, human MPO (30 nM) was addedto reaction mixtures. Data represent the mean±SD of triplicatedeterminations. Each bar within a cluster for a given conditionrepresents results obtained from independent experiments performed withneutrophil preparations from a distinct donor. PMN(MPO+), neutrophilsisolated from normal subjects; PMN(MPO−), neutrophils isolated fromMPO-deficient subjects.

FIG. 5. Characterization of neutrophil-dependent initiation of lipidperoxidation of endogenous plasma lipids. Neutrophils (1×10⁶/ml)isolated from normal subjects (PMN) were incubated at 37° C. in HBSSsupplemented with DTPA (100 μM, pH 7.0) and fresh human plasma (50%v/v). Cells were activated by addition of phorbol myristate acetate(PMA, 200 nM) and then incubated for 2 h (Complete System). The contentof 9-H(P)ODE and 9-H(P)ETE formed within endogenous plasma lipids werethen determined by LC/ESI/MS/MS. Additions or deletions to the CompleteSystem were as indicated. The final concentrations of additions to theComplete System were 30 nM human MPO, 1 mM NaN₃, 300 nM catalase (Cat),300 nM heat inactivated-catalase (hiCat), 100 μM methionine (Met), 100μM ascorbate and 10 μg/ml superoxide dismutase (SOD). Data represent themean±SD of three independent experiments.

FIG. 6. Characterization of MPO-dependent initiation of lipidperoxidation of endogenous plasma lipids. Fresh human plasma (50%, v/v)was incubated with isolated human MPO (30 nM) at 37° C. in HBSSsupplemented with DTPA (100 μM, pH 7.0) and a H₂O₂-generating systemcomprised of glucose/glucose oxidase (G/GO) for 12 h (Complete System).Under this condition, a continuous flux of H₂O₂ is formed at 10 μM/hr.The content of 9-H(P)ODE and 9-H(P)ETE formed within endogenous plasmalipids were then determined by LC/ESI/MS/MS. Additions or deletions tothe Complete System were as indicated. The final concentrations ofadditions to the Complete System were 1 mM NaN₃, 300 nM catalase (Cat),300 nM heat-inactivated catalase (hiCat), 200 nM SOD, 100 μM methionine(Met), and 100 μM ascorbate. Data represent the mean±SD of threeindependent experiments.

FIG. 7. Oxidized phosphatidyl choline species generated by MPO oxidationof LDL are enriched in atherosclerotic lesions. The contents of theindicated oxidized PC species were determined in native LDL and LDLoxidized by the MPO-H₂O₂—NO₂ system (NO₂-LDL) using LC/ESI/MS/MS. Datarepresent the mean±S.D. of triplicate determinations of a representativeexperiment performed two times. The content of PAPC in LDL and NO₂-LDLpreparations were 0.122±0.07 and 0.008±0.001 μmol/mg apoprotein,respectively. The content of PLPC in LDL and NO₂-LDL preparations were0.88±0.05 and 0.35±0.05 μmol/mg apoprotein, respectively. The thoracicaorta from Watanabe Heritable Hyperlipidemic Rabbits was isolated,rinsed in Argon sparged PBS supplemented with 100 μM BHT and 100 μMDTPA, submerged in the same buffer, covered in argon, flash-frozen inliquid nitrogen and then stored at −80° C. until analysis. Aortaerelatively free of lipid lesions were obtained from WHHL rabbits age10-12 weeks, while aortae with confluent lesions were recovered fromWHHL rabbits >6 months old. Individual frozen aortae were pulverizedwith stainless steel mortar and pestle under liquid nitrogen, the powdertransferred to glass screw capped test tubes equipped with PTFE-linedcaps, and then lipids were extracted by the method of Bligh and Dyerunder Argon in the presence of BHT. Three aortae were analyzed in eachgroup. Quantification of lipids was then performed by LC/ESI/MS/MS. Dataare expressed as mean±S.D.

FIG. 8. Content of select MPO-generated oxidized lipids inatherosclerotic plague material of human patients and normal aorticintima of heart transplant donors.

FIG. 9. The content of MPO in isolated leukocytes (Leukocyte-MPO) andper ml of blood (Blood-MPO) were determined in 333 subjects (158 withknown coronary artery disease and 175 without angiographicallysignificant CAD) as described under “Methods.” Box-whisker plots of MPOlevels vs. CAD status are shown. Boxes encompass the 25^(th) to 75^(th)percentiles. Lines within boxes represent median values. Bars representthe 2.5^(th) and 97.5^(th) percentiles. ANC, absolute neutrophil count;CAD, coronary artery disease; PMN, polymorphonuclear leukocyte.

FIG. 10. Model 1—Odds ratios adjusted for risk factors significantfollowing univariate adjustment: age, gender, hypertension, smokinghistory, HDLc, WBC quartile and MPO quartile. Model 2—Odds ratiosadjusted for Framingham Global Risk assessment, WBC and MPO quartile.Closed circles, unadjusted odd ratios. Closed triangles, Model 1. Closedsquares, Model 2.

FIG. 11. Cytogram of WBC from an individual whose MPO level perneutrophil is below the average in a population (left panel), and anindividual whose MPO level per neutrophil is above average in apopulation (right panel).

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are specifically incorporated herein byreference.

The present invention provides diagnostic tests for characterizing anindividual's risk for developing or having CVD. In one aspect, themethod comprises obtaining the level of MPO activity in a bodily sampleobtained from the individual. In another aspect, the method comprisesobtaining the level of MPO mass in a bodily sample from the individual.In another aspect, the method comprises obtaining the level of one ormore select MPO-generated oxidation products in a bodily sample from theindividual or test subject. Such MPO-generated oxidation products areselected from the group consisting of dityrosine, nitrotyrosine,methionine sulphoxide and a lipid peroxidation product. In yet anotheraspect, the method comprises obtaining the level of MPO activity, or MPOmass, or both, and the level of one or more select MPO-generatedoxidation products in a bodily sample obtained from the individual.

The level of MPO activity or MPO mass or select MPO-generated oxidationproduct in the individual's bodily sample is then compared to apredetermined value to provide a risk value which characterizes theindividual's risk of developing or having CVD.

The present invention also relates to kits that comprise assays for MPOactivity or mass, or the select MPO-generated oxidation product. Suchassays have appropriate sensitivity with respect to predetermined valuesselected on the basis of the present diagnostic tests. The present kitsdiffer from those presently commercially available for MPO by including,for example, different cut-offs, different sensitivities at particularcut-offs, as well as instructions or other printed material forcharacterizing risk based upon the outcome of the assay.

Preparation of Bodily Sample

Whole blood is obtained from the individual or test subject usingstandard clinical procedures. Plasma is obtained from whole bloodsamples by centrifugation of anti-coagulated blood. Such processprovides a buffy coat of white cell components and a supernatant of theplasma.

Serum is collected by centrifugation of whole blood samples that havebeen collected in tubes that are free of anti-coagulant. The blood ispermitted to clot prior to centrifugation. The yellowish-reddish fluidthat is obtained by centrifugation is the serum.

Leukocytes can be isolated from whole blood samples by any of varioustechniques including buoyant density centrifugation as described in theexamples below.

Myeloperoxidase and Myeloperoxidase-Generated Oxidation Products

MPO (donor: hydrogen peroxide, oxidoreductase, EC 1.11.1.7) is atetrameric, heavily glycosylated, basic (PI. 10) heme protein ofapproximately 150 kDa. It is comprised of two identical disulfide-linkedprotomers, each of which possesses a protoporphyrin-containing 59-64 kDaheavy subunit and a 14 kDa light subunit (Nauseef, W. M, et al., Blood67:1504-1507; 1986.)

MPO is abundant in neutrophils and monocytes, accounting for 5%, and 1to 2%, respectively, of the dry weight of these cells (Nauseef, W. M, etal., Blood 67:1504-1507; 1986, (Hurst, J. K. In: Everse J.; Everse K.;Grisham M. B., eds. Peroxidases in chemistry and biology 1st ed. BocaRaton: CRC Press; 1991:37-62.) The heme protein is stored in primaryazurophilic granules of leukocytes and secreted into both theextracellular milieu and the phagolysosomal compartment followingphagocyte activation by a variety of agonists (Klebanoff, S. J, et al.The neutrophil: functions and clinical disorders. Amsterdam: ElsevierScientific Publishing Co.; 1978.) Immunohistochemical methods havedemonstrated that MPO is present in human atheroscloerotic lesions.However, MPO has not yet been shown to be present at increased levels inblood samples from individuals with atherosclerosis.

A recently proposed working kinetic model for MPO is shown in FIG. 1.MPO is a complex heme protein which possesses multiple intermediatestates, each of which are influenced by the availability of reducedoxygen species such as O₂ ⁻ and H₂O₂, and nitric oxide (NO, nitrogenmonoxide) (Abu-Soud, H. M., et al., J. Biol. Chem. 275:5425-5430; 2000).At ground state, MPO exists in the ferric (Fe(III)) form. Upon additionof H₂O₂, the heme group of MPO is oxidized two e⁻ equivalents forming areactive ferryl π cation radical intermediate termed Compound I. In thepresence of halides such as Cl⁻, Br⁻, and I⁻, and the psuedohalidethiocyanate (SCN⁻), Compound I is readily reduced in a single two e⁻step, regenerating MPO-Fe(III) and the corresponding hypohalous acid(HOX). At plasma levels of halides and thiocyanate (100 mM Cl⁻, 100 mMBr⁻ 50 mM SCN⁻, 100 nM I⁻, chloride is a preferred substrate andhypochlorous acid (HOCl), a potent chlorinating oxidant, is formed(Foote, C. S., et al; Nature 301:715-726; 1983, Weiss, S. J., et al. J.Clin. Invest. 70:598-607; 1982).

Compound I can also oxidize numerous organic substrates while the hemeundergoes two sequential one e⁻ reduction steps, generating compound IIand MPO-Fe(III), respectively (FIG. 1). Low molecular weight compoundsprimarily serve as substrates for MPO, generating diffusible oxidantsand free radical species which can then convey the oxidizing potentialof the heme to distant targets. In addition to halides and SCN⁻, some ofthe naturally occurring substrates for MPO include nitrite (NO₂ ⁻) (vander Vliet, A., et al., J Biol. Chem. 272:7617-7625; 1997), tyrosine (vander Vliet, A., et al., J. Biol. Chem. 272:7617-7625; 1997), ascorbate(Marquez, L. A., et al., J. Biol. Chem. 265:5666-5670; 1990), urate(Maehly, H. C. Methods Enzymol. 2:798-801; 1955), catecholamines(Metodiewa, D., et al., Eur. J. Biochem. 193:445-448; 1990), estrogens(Klebanoff, S. J. J. Exp. Med 145:983-998; 1977), and serotonin(Svensson, B. E. Chem. Biol. Interact. 70:305-321; 1989). MPO-Fe(III)can also be reduced to an inactive ferrous form, MPO-Fe(II) (Hurst, J.K. In: Everse J.; Everse K.; Grisham M. B., eds. Peroxidases inchemistry and biology 1st ed. Boca Raton: CRC Press; 1991:37-62,(Kettle, A. J., et al., Redox. Rep. 3:3-15; 1997). MPO-Fe(III) andMPO-Fe(II) bind to O₂ ^(•−), and O₂, respectively, forming a ferrousdioxy intermediate, compound III (MPO-Fe(II)—O₂) (FIG. 1). Spectralstudies demonstrate that addition of H₂O₂ to Compound III ultimatelyforms compound II. Thus, compound III may indirectly promote one e⁻peroxidation reactions.

Recent studies identify a role for NO, a relatively long-lived freeradical generated by nitric oxide synthase (NOS), in modulating MPOperoxidase activity (Abu-Soud, H. M., et al., J. Biol. Chem.275:5425-5430; 2000). MPO and the inducible isoform of NOS arecolocalized in the primary granule of leukocytes. During phagocyteactivation, such as during ingestion of bacteria, MPO and NOS aresecreted into the phagolysosome and extracellular compartments, andnitration of bacterial proteins is observed (Evans, T. J., et al., Proc.Natl. Acad. Sci. USA 93:9553-9558; 1996). Rapid kinetics studiesdemonstrate that at low levels of NO, the initial rate of MPO-catalyzedperoxidation of substrates is enhanced. The mechanism is throughacceleration of the rate-limiting step in MPO catalysis, reduction ofcompound I to MPO-Fe(III) (FIG. 1) (Abu-Soud, H. M., et al., J. Biol.Chem. 275:5425-5430; 2000, Abu-Soud, H. M., et al. Nitric oxide is aphysiological substrate for mammalian animal peroxidases. Submitted;2000). At higher levels of NO, reversible inhibition of MPO occursthrough formation of a spectroscopically distinguishable nitrosylcomplex, MPO-Fe(III)-NO (Abu-Soud, H. M., et al., J. Biol. Chem.275:5425-5430; 2000). NO also can serve as a substrate for MPO compoundI, resulting in its reduction to Compound II (Abu-Soud, H. M., et al.Nitric oxide is a physiological substrate for mammalian animalperoxidases. Submitted; 2000). Furthermore, in the presence of NO, theoverall turnover rate of MPO through the peroxidase cycle is enhancednearly 1000-fold (Abu-Soud, H. M., et al. Nitric oxide is aphysiological substrate for mammalian animal peroxidases. Submitted;2000). Finally, NO also reversibly binds to MPO-Fe(II) forming thecorresponding MPO-Fe(II)-NO intermediate, which is in equilibrium withMPO-Fe(II) and MPO-Fe(III)-NO (FIG. 1) (Abu-Soud, H. M., et al., J.Biol. Chem. 275:5425-5430; 2000, Abu-Soud, H. M., et al. Nitric oxide isa physiological substrate for mammalian animal peroxidases. Submitted;2000).

As described above, MPO can utilize a variety of cosubstrates with H₂O₂to generate reactive oxidants as intermediates. Many stable end-productsgenerated by these species have been characterized and shown to beenriched in proteins, lipids, and LDL recovered from humanatherosclerotic lesions (Chisolm, G. M., et al., Proc. Natl. Acad. Sci.USA 91:11452-11456; 1994, Hazell, L. J., et al, J. Clin. Invest.97:1535-1544; 1996, Hazen, S. L., et al., J. Clin. Invest. 99:2075-2081;1997, Leeuwenburgh, C., et al, J. Biol. Chem. 272:1433-1436; 1997,Leeuwenburgh, C., et al., J. Biol. Chem. 272:3520-3526; 1997). FIG. 2summarizes some of the reactive intermediates and products formed byMPO, any of which are known to be enriched in vascular lesions.

Methods of Determining MPO Activity

Myeloperoxidase activity may be determined by any of a variety ofstandard methods known in the art. One such method is acolorimetric-based assay where a chromophore that serves as a substratefor the peroxidase generates a product with a characteristic wavelengthwhich may be followed by any of various spectroscopic methods includingUV-visible or fluorescence detection. Additional details of colorimetricbased assays can be found in Kettle, A. J. and Winterbourn, C. C. (1994)Methods in Enzymology. 233: 502-512; and Klebanoff, S. J., Waltersdorph,A. N. and Rosen, H. (1984) Methods in Enzymology. 105: 399-403, both ofwhich are incorporated herein by reference. An article by Gerber,Claudia, E. et al, entitled “Phagocytic Activity and Oxidative Burst ofGranulocytes in Persons with Myeloperoxidase Deficiency” published in1996 in Eur. J. Clin. Chem Clin Biochem 34:901-908, describes a methodfor isolation for polymorphonuclear leukocytes (i.e. neutrophils) andmeasurement of myeloperoxidase activity with a colorometric assay, whichinvolves oxidation of the chromgen 4-chloro-1-naphthol.

Peroxidase activity may be determined by in situ peroxidase staining inMPO containing cells with flow cytometry-based methods. Such methodsallow for high through-put screening for peroxidase activitydeterminations in leukocytes and subpopulations of leukocytes. Anexample is the cytochemical peroxidase staining used for generatingwhite blood cell count and differentials with hematology analyzers basedupon peroxidase staining methods. For example, the Advia 120 hematologysystem by Bayer analyzes whole blood by flow cytometry and performsperoxidase staining of white blood cells to obtain a total white bloodcell count (CBC) and to differentiate amongst the various white bloodcell groups.

With these methods, whole blood enters the instrument and red bloodcells are lysed in a lysis chamber. The remaining white blood cells arethen fixed and stained in situ for peroxidase activity. The stainedcells are channeled into the flow cytometer for characterization basedupon the intensity of peroxidase staining and the overall size of thecell, which is reflected in the amount of light scatter of a given cell.These two parameters are plotted on the x and y axis, respectively, byconventional flow cytometry software, and clusters of individual cellpopulations are readily discernible. These include, but are not limited,to neutrophils, monocytes and eosinophils, the three major leukocytepopulations containing visible peroxidase staining.

During the course of these analyses, leukocytes such as monocytes,neutrophils, eosinophils and lymphocytes are identified by the intensityof peroxidase staining and their overall size. Information about theoverall peroxidase activity staining within specific cell populations isthus inherent in the position of individual cell clusters (e.g.neutrophil, monocyte, eosinophil clusters) and peroxidase levels withinspecific cell populations may be determined. Peroxidaseactivity/staining in this detection method is compared to a peroxidasestain reference or calibrant. Individuals with higher levels ofperoxidase activity per leukocyte are identified by having a cellpopulation whose location on the cytogram indicates higher levels ofperoxidase (i.e., average peroxidase activity per leukocyte) or bydemonstrating a sub-population of cells within a cell cluster (e.g.neutrophil, monocyte, eosinophil clusters) which contain higher levelsof peroxidase activity either on average or in a higher subgroup, suchas the higher tertile or quartile.

Methods of Determining MPO Mass

The mass of myeloperoxidase in a given sample is readily determined byan immunological method, e.g. ELISA. Commercial kits for MPOquantification by ELISA are available.

MPO mass in a sample can also be determined indirectly by in situperoxidase staining of the bodily sample. Methods which analyzeleukocyte peroxidase staining can be performed on whole blood, such asthose with hematology analyzers which function based upon in situperoxidase staining. Previous studies by other investigators havedemonstrated that the overall intensity of staining is proportional toperoxidase mass (e.g. Claudia E. Gerber, Selim Kuci, Matthias Zipfel,Ditrich Niethammer and Gernot Bruchfelt, “Phagocytic activity andphagocytic activity and oxidative burst of granulocytes in persons withmyeloperoxidase deficiency” European Journal of Clinical Chemistry andClinic Biochemistry (1996) 34: 901-908).

Flow cytometry through a hematology analyzer is a high through-puttechnique for quantifying the parameters used in determining MPOactivity or mass levels or numbers of cells containing elevated levelsof MPO activity or mass. The advantage of using such a technique is itsease of use and speed. The Advia 120 can perform 120 complete cell bloodcount and differentials in one hour and utilizes only a few microlitersof blood at a time. All the data necessary for determination of theperoxidase activity is held within the flow cytometry cell clusters usedto ultimately calculate the total white blood cell count anddifferential. With minor adjustments to software of this apparatus, thereadout can be modified to include multiple different indices of overallperoxidase activity. For example, individuals whose neutrophil clusterscontain an overall increase in the average peroxidase activity (i.e.increased mean peroxidase index) will be at increased risk fordevelopment of cardiovascular disease. In addition to simply determiningthe mean peroxidase activity for a given cell type, individuals atincreased risk of developing CVD can be identified by examining theoverall distribution of peroxidase activity within a given cell cluster(mean+mode, etc). It is expected that by looking at the population ofperoxidase activity per leukocyte, individuals who possess leukocyteswith a higher proportion of cells containing a high peroxidase activityin a subset of cells (for example, the upper quartile, or the uppertertile) may be at particularly high risk.

Levels of MPO Activity and MPO Mass

The level of MPO activity or MPO mass in the body fluid can bedetermined by measuring the MPO activity or MPO mass in the body fluidand normalizing this value to obtain the MPO activity or mass per ml ofblood, per ml of serum, per ml of plasma, per leukocyte (e.g. neutrophilor monocyte), per weight, e.g. mg of total blood protein, per weight ofleukocyte protein (e.g. per weight of neutrophil or monocyte protein).Alternatively, the level of MPO activity or MPO mass in the body fluidcan be a representative value which is based on MPO activity in the testsubjects blood or blood derivatives. For example the level of MPOactivity can be the percentage or the actual number of the testsubject's neutrophils or monocytes that contain elevated levels of MPOactivity or MPO mass. Examples of other representative values include,but are not limited to, arbitrary units for a parameter that can beobtained from a flow cytometry based cytogram, such as the position ofthe neutrophil cluster on the X and Y axes, or the angle of the majoraxis of the neutrophil cluster relative to the X and Y axes.

Myeloperoxidase-Generated Oxidation Products Role of MPO in theGeneration of HETEs and HODEs and Oxidized Cholesterol Esters

A role for MPO in the oxidation of LDL and the initiation of lipidperoxidation has recently been questioned by several investigators.Noguchi and colleagues examined the capacity of leukocytes isolated fromwild-type and MPO knockout mice to promote oxidation of LDL in modelsystems ex vivo and observed only modest differences in the parametersof lipid oxidation monitored. (Noguchi N, et al. J. Biochem. (Tokyo)2000; 127:971-976). It has also recently been suggested thatMPO-catalyzed oxidation of LDL is inhibited, rather than promoted, bythe presence of NO₂ ⁻, particularly when focusing upon protein oxidationproducts. (Carr A C, et al, J. Biol. Chem. 2001; 276:1822-1828).Moreover, an antioxidant rather than a pro-oxidant function forMPO-generated tyrosine oxidation products and LDL oxidation has beenproposed. (Santanam N., et al. J. Clin. Invest 1995; 95:2594-2600, ExnerM. et al., FEBS Lett. 2001; 490:28-31). It has also been suggested bysome investigators that HOCl generated by MPO can promote oxidation oflipoprotein lipids and formation of hydroperoxides (Panasenko O M.,Biofactors 1997; 6:181-190), whereas other studies have not supportedthese observations. (Schmitt D, et al., Biochem. 1999; 38:16904-16915,Hazen S L, et al., Circ. Res. 1999; 85:950-958). Finally, recent studieshave noted species differences between murine and human leukocytes withrespect to MPO and generation of reactive oxidant species. (Xie Q W, etal., Biological oxidants: generation and injurious consequences. SanDiego, Calif., USA, Academic Press, 1992, Rausch P G, et al., Blood1975; 46:913-919, Nauseef W M., J. Clin. Invest 2001; 107:401-403,Brennan M L, et al. J. Clin. Invest 2001; 107:419-430).

To determine the role of MPO in promoting lipid oxidation in plasma, weincubated activated neutrophils from healthy subjects and subjects witha myeloperoxidase deficiency with whole plasma (50%, v/v) andphysiological levels of Cl⁻ (100 mM final). Phagocytes were activatedwith PMA and the formation of specific oxidation products of linoleicand arachidonic acids, respectively, was determined by LC/ESI/MS/MS.

MPO and Lipoprotein Isolation

MPO (donor: hydrogen peroxide, oxidoreductase, EC 1.11.1.7) was isolatedand characterized as described. (Heinecke J W, et al., J. Biol. Chem.1993; 268:4069-4077, Wu W, et al., Biochemistry 1999; 38:3538-3548).Purity of isolated MPO was established by demonstrating a R/Z≧0.85(A₄₃₀/A₂₈₀), SDS PAGE analysis with Coomassie Blue staining, and in-geltetramethylbenzidine peroxidase staining to confirm no eosinophilperoxidase contamination. (Wu W, et al., Biochemistry 1999;38:3538-3548). Purified MPO was stored in 50% glycerol at −20° C. Enzymeconcentration was determined spectrophotometrically (ε₄₃₀=170,000M⁻¹cm⁻¹). (Odajima T, et al. Biochim. Biophys. Acta. 1970; 71-77). LDLwas isolated from fresh plasma by sequential ultracentrifugation as a1.019<d<1.063 g/ml fraction with dialysis performed in sealed jars underargon atmosphere. (Hatch F T. Adv. Lipid Res. 1968; 6:1-68). Finalpreparations were kept in 50 mM sodium phosphate (pH 7.0), 100 μM DTPAand stored under N₂ until use. LDL concentrations are expressed per mgof LDL protein.

Human Neutrophil Preparations

Human neutrophils were isolated from whole blood obtained from normaland MPO-deficient subjects, as described. (Hazen S L, et al., J. Biol.Chem. 1996; 271:1861-1867). Neutrophils preparations were suspended inHBSS (Mg²⁺-, Ca²⁺-, phenol- and bicarbonate-free, pH 7.0) and usedimmediately for experiments.

Lipid Peroxidation Reaction

Isolated human neutrophils (10⁶/ml) were incubated at 37° C. with either50% (v/v) normal human plasma or isolated human LDL (0.2 mg/ml) underair in HBSS supplemented with 100 μM DTPA. Neutrophils were activated byadding 200 nM phorbol myristate acetate (PMA) and maintained insuspension by gentle mixing every 5 min. After 2 h, reactions werestopped by immersion in ice/water bath, centrifugation at 4° C. andimmediate addition of 50 μM butylated hydroxytoluene (BHT) and 300 nMcatalase to the supernatant. Lipid peroxidation products in thesupernatant were then rapidly assayed as described below.

Reactions with isolated MPO were typically performed at 37° C. in sodiumphosphate buffer (20 mM, pH 7.0) supplemented with 100 μM DTPA using 30nM MPO, 1 mM glucose (G), 20 ng/ml glucose oxidase (GO). Under thiscondition, a constant flux of H₂O₂ (0.18 μM/min) was generated by theglucose/glucose oxidase (G/GO) system. Unless otherwise stated,reactions were terminated by immersion in ice/water bath and addition ofboth 50 μM BHT and 300 nM catalase to the reaction mixture.

Lipid Extraction and Sample Preparation

Lipids were extracted and prepared for mass spectrometry analysis underargon or nitrogen atmosphere at all steps. First, hydroperoxides in thereaction mixture were reduced to their corresponding hydroxides byadding SnCl₂ (1 mM final). A known amount of deuterated internalstandard,12(S)-hydroxy-5,8,10,14-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid(12-HETE-d8; Cayman Chemical Company, Ann Arbor, Mich.) was added to thesample, and then plasma lipids were extracted by adding a mixture of 1 Macetic acid/2-isopropanol/hexane (2/20/30, v/v/v) at a ratio of 5 mlorganic solvent mix:1 ml plasma. Following vortexing of the mixture andcentrifugation, lipids were extracted into the hexane layer. Plasma wasre-extracted by addition of an equal volume of hexane, followed byvortexing and centrifugation. Cholesteryl ester hydroperoxides(CE-H(P)ODEs) were analyzed as their stable SnCl₂-reduced hydroxideforms by drying of the combined hexane extracts under N₂, reconstitutingsamples with 200 μl 2-isopropanol/acetonitrile/water (44/54/2, v/v/v)and storage at −80° C. under argon until analysis. For the assay of freefatty acids and their oxidation products, total lipids (phospholipids,cholesterol esters, triglycerides) were dried under N₂, re-suspended in1.5 ml 2-isopropanol and then fatty acids were released by basehydrolysis with 1.5 ml 1M NaOH at 60° C. for 30 min under argon. Thehydrolyzed samples were acidified to pH 3.0 with 2M HCl and fatty acidswere extracted twice with 5 ml hexane. The combined hexane layers weredried under N₂, resuspended in 100 μl methanol and stored under argon at−80° C. until analysis by LC/ESI/MS/MS), as described below.

HPLC Fractionation of Plasma Filtrate

In order to study the role played by low molecular weight compounds inplasma as substrates for MPO in promotion of lipid peroxidation, wholeplasma from normal healthy donors was filtered through a 10 kDa MWt cutoff filter (Centriprep YM-10, Millipore-Corporation Bedford, Mass. USA)by centrifugation. The filtrate of plasma was used either directly orfollowing fractionation by HPLC. Reverse phase HPLC fractionation of wasperformed using a Beckman C-18 column (4.6×250 mm, 5 μm ODS; BeckmanInstruments, Inc. Fullerton, Calif.). The separation of low molecularweight compounds in plasma filtrate (0.5 ml) was carried out at the flowrate 1.0 ml/min with the following gradient: 100% mobile phase A (watercontaining 0.1% acetic acid) over 10 min, then linear gradient to 100%mobile phase B (methanol containing 0.1% acetic acid) over 10 min,followed by 100% mobile phase B over 5 min. Effluent was collected as 1ml fractions, dried under N₂, and then resuspended in buffer (0.1 ml)for analysis. Fractionation of plasma filtrate (0.5 ml) by strong anionexchange HPLC (SAX-HPLC) was performed on a SPHERIS HPLC column (4.6×250mm, 5 μm SAX; Phase Separations Inc. Norwalk Conn.). The separation oflow molecular weight compounds in plasma filtrate was carried out at theflow rate 0.9 ml/min under isocratic conditions using 45 mM ammoniumacetate buffer (pH 4.0) as mobile phase. Effluent was collected as 1.0ml fractions, dried under N₂, and then resuspended in buffer (0.1 ml)for analysis.

a) Mass Spectrometry

LC/ESI/MS/MS was employed to quantify free radical-dependent oxidationproducts of arachidonic acid (9-hydroxy-5,7,11,14-eicosatetraenoic acidand 9-hydroperoxy-5,7,11,14-eicosatetraenoic acid (9-H(P)ETE)), andlinoleic acid (9-hydroxy-10,12-octadecadienoic acid and9-hydroperoxy-10,12-octadecadienoic acid (9-H(P)ODE)). Immediately priorto analysis, one volume of H₂O was added to five volumesmethanol-suspended sample, which was then passed through a 0.22 μmfilter (Millipore Corporation, Bedford, Mass.). Sample (20 μl) wasinjected onto a Prodigy C-18 column (1×250 mm, 5 μm ODS, 100 A;Phenomenex, Rancho Palos Verdes, Calif.) at a flow rate of 50 μl/min.The separation was performed under isocratic conditions using 95%methanol in water as the mobile phase. In each analysis, the entirety ofthe HPLC column effluent was introduced onto a Quattro II triplequandrupole MS (Micromass, Inc.). Analyses were performed usingelectrospray ionization in negative-ion mode with multiple reactionmonitoring (MRM) of parent and characteristic daughter ions specific forthe isomers monitored. The transitions monitored were mass-to-chargeratio (m/z) 295 171 for 9-HODE; m/z 319 151 for 9-HETE; m/z 327 184 for12-HETE-d8. N₂ was used as the curtain gas in the electrosprayinterface. The internal standard 12-HETE-d8 was used to calculateextraction efficiencies (which were >80% for all analyses). Externalcalibration curves constructed with authentic standards were used toquantify 9-HETE and 9-HODE.

b) RP-HPLC Quantification of CE-H(P)ODEs

Sample (100 μl) reconstituted in methanol (without base hydrolysis) wereinjected onto a Beckman C-18 column (4.6×250 mm, 5 μm ODS; BeckmanInstruments, Inc., Fullerton, Calif.). Lipids were separated using anisocratic solvent system comprised of 2-isopropanol/acetonitrile/water(44/54/2, v/v/v) at a flow rate of 1.5 ml/min. CE-H(P)ODEs werequantified as their stable hydroxide forms by UV detection at 234 nmusing CE-9-HODE (Cayman Chemical Company, Ann Arbor, Mich.) forgeneration of an external calibration curve.

Results

Normal neutrophils generated significant levels of 9-H(P)ODE and9-(H)PETE in plasma following cell activation by PMA (FIG. 4). In starkcontrast, MPO-deficient neutrophils failed to generate significantlevels of lipid peroxidation products following stimulation with PMA,despite their enhanced capacity to produce O

. Addition of catalytic amounts of MPO restored the capacity ofMPO-deficient neutrophils to initiate peroxidation of endogenous plasmalipids (FIG. 4).

Addition of catalase, but not heat inactivated catalase, to cellmixtures resulted in the near complete ablation of lipid peroxidation inplasma, strongly suggesting a critical role for H₂O₂ in thecell-dependent reaction (FIG. 5). Incubation of reaction mixtures withsuperoxide dismutase (SOD) failed to attenuate oxidation of plasmalipids (FIG. 5). In contrast, addition of heme poisons (e.g. azide,cyanide) and the water-soluble antioxidant ascorbate resulted incomplete inhibition of neutrophil-depended peroxidation of plasmalipids. Finally, addition of HOCl scavengers such as dithiothreitol andthe thioether methionine, failed to attenuate neutrophil-dependentperoxidation of endogenous plasma lipids, assessed by quantification of9-H(P)ODE and 9-H(P)ETE (FIG. 5).

Results thus far presented strongly suggest that neutrophils employ theMPO-H₂O₂ system to generate reactive species distinct from chlorinatingintermediates as the primary oxidants for initiation of lipidperoxidation in plasma. To confirm a physiological role for MPO, we nextadded purified human MPO and a H₂O₂-generating system (glucose/glucoseoxidase, G/GO) to plasma and monitored formation of specific oxidationproducts by LC/ESI/MS/MS analysis. Formation of 9-H(P)ODE and 9-H(P)ETEoccurred readily and had an absolute requirement for the presence ofboth MPO and the H₂O₂-generating system (FIG. 6). Lipid oxidation wasagain inhibited by catalase, azide or ascorbate, but was not affected byaddition of SOD or methionine (FIG. 6). Collectively, these resultsstrongly support a pivotal role for the MPO-H₂O₂ system of leukocytes asa primary mechanism for initiating lipid peroxidation in complexbiological tissues and fluids such as plasma.

MPO Oxidation of LDL and the Presence of the Resultant OxidationProducts in Atherosclerotic Lesions General Procedures.

Human myeloperoxidase (donor: hydrogen peroxide, oxidoreductase, EC1.11.1.7) and LDL were isolated and quantified as described (Podrez, E.A, et al., 1999, J. Clin. Invest. 103:1547). All buffers were treatedwith Chelex-100 resin (Bio-Rad, Hercules, Calif.) and supplemented withdiethylenetriaminepentaacetic acid (DTPA) to remove trace levels oftransition metal ions that might catalyze LDL oxidation duringincubations. LDL was labeled with Na[¹²⁵I] to a specific activitybetween 100 and 250 dpm/ng protein, as described (Hoppe, G., et al.,1994, J. Clin. Invest. 94, 1506-12). Extraction of cellular lipids andthin-layer chromatography separation of radiolabeled cholesterol estersand free cholesterol were performed as described (Podrez, E. A, et al.,1999, J. Clin. Invest. 103:1547). Incorporation of [¹⁴C]oleate intocholesteryl esters by cells following incubation with the indicatedlipoproteins (50 μg/ml), were determined as described (Podrez, E. A, etal., 1999, J. Clin. Invest. 103:1547). Rabbit thoracic aortae wereisolated from WHHL Rabbits, rinsed in argon-sparged PBS supplementedwith 100 μM butylated hydroxytoluene (BHT) and 100 μM DTPA, submerged inthe same buffer, covered in argon and flash frozen in liquid nitrogenand then stored at −80° C. until analysis. Aortae relatively free oflipid lesions were obtained from WHHL rabbits age 10-12 weeks, whileaortae full of lesions were recovered from WHHL rabbits greater than 6months old.

Lipoprotein Modification.

LDL modified by MPO-generated nitrating intermediates (NO₂-LDL) wasformed by incubating LDL (0.2 mg protein/ml) at 37° C. in 50 mM sodiumphosphate, pH 7.0, 100 μM DTPA, 30 nM MPO, 100 μg/ml glucose, 20 ng/mlglucose oxidase and 0.5 mM NaNO₂ for 8 h unless otherwise specified.Under these conditions, a constant flux of H₂O₂ (10 μM/hr) is generatedby the glucose/glucose oxidase system, as determined by the oxidation ofFe(II) and formation of Fe(III)-thiocyanate complex (van der Vliet, A.,et al., 1997, J. Biol. Chem., 272:7617). Oxidation reactions wereterminated by addition of 40 μM BHT and 300 nM catalase to the reactionmixture. LDL acetylation was performed as described earlier (Podrez, E.A, et al., 1999, J. Clin. Invest. 103:1547).

Phospholipid Separation and Mass Spectrometric Analysis.

Lipids were maintained under inert atmosphere (argon or nitrogen) at alltimes. Lipids from either oxidized PAPC or PLPC vesicles, or fromNO₂-LDL, were extracted three times sequentially by the method of Blighand Dyer [Bligh, 1959 #52] immediately after adding an equal volume ofsaturated NaCl solution (to enhance lipid extraction). The combinedchloroform extracts were evaporated under nitrogen, and lipids were thenresuspended in methanol (at approximately 200 μg/0.1 mL), filteredthrough an Acrodisc CR PTFE filter and applied on a reverse-phase column(Luna C18, 250×10 mm, 5 μm, Phenomenex, Torrence, Calif., USA). Lipidswere resolved at a flow rate of 3 mL/min using a ternary(acetonitrile/methanol/H₂O) gradient generated by a Waters 600 EMultisolvent delivery system HPLC (Waters, Milford, Mass., USA), andmonitored using an evaporative light scattering detector (Sedex 55,Sedere, Alfortville, France).

Further fractionation and isolation of bioactive lipids was performed oncombined lipid extracts from three separations that were dried under N₂,resuspended in chloroform (300 μl) supplemented with BHT and maintainedunder argon atmosphere. An aliquot of the fraction (⅔rds) was removed,evaporated under nitrogen and resuspended in HPLC buffer(methanol/water; 85/15; v/v) immediately prior to injection on reversephase HPLC column.

Mass spectrometric analyses were performed on a Quatro IItriple-quadrupole mass spectrometer (Micromass, Inc., Altrincham, U.K.)equipped with an electrospray ionization (ESI) probe and interfaced withan HP 1100 HPLC (Hewlett-Packard, Wilmington, Del.). Lipids (both freeand following derivatization) were resolved on a Luna C18 250×4.6 mm, 5μm column (Phenomenex, Torrance, Calif.) at a flow rate of 0.8 ml/min. Adiscontinuous gradient (Gradient II) was used by mixing solvent A(methanol (MeOH):H₂O, 85:15, v:v) with solvent B (MeOH), as follows:isocratic elution with solvent A from 0-7 min; increasing to 88% solventB from 7-10 min; increasing to 91% solvent B from 10-34 min; and thenincreasing to 94% solvent B from 34-52 min). The column effluent wassplit such that 45 μl/min was introduced to the mass spectrometer and755 μl/min was collected and analyzed for biological activity. In somecases, biological activity was also determined using the same gradientfollowing injection of authentic standards. Mass spectrometric analyseswere performed on-line using electrospray ionization tandem massspectrometry (ESI/MS/MS) in the positive ion mode with multiple reactionmonitoring (MRM) mode (cone potential 60 eV/collision energy 20-25 eV).The MRM transitions used to detect the oxidized phosphopholipids presentin each fraction were the mass to charge ratio (m/z) for the molecularcation [MH]⁺ and the daughter ion m/z 184, the phosphocholine group(i.e. [MH]⁺→m/z 184). Oxime derivatives of phospholipids were monitoredat m/z [MH+29]⁺→m/z 184.

Quantification of the various oxidized PC species was performed usingLC/ESI/MS/MS in positive ion mode using MRM. Formic acid (0.1%) wasincluded in the mobile phases. Distinct oxidized phospholipid specieswere identified by using m/z for protonated parent→daughter transitionsspecific for each individual phospholipid and their retention times, asillustrated in FIGS. 2 and 3. OV-PC and ND-PC were quantified similarlybut by also monitoring at the m/z for the transition between thehemiacetal formed with methanol for each analyte and the loss of polarhead group (m/z 184).

Lipids were initially extracted three times by the method of Bligh andDyer (Bligh, E. G., et al., 1959, Canadian Journal of BiochemicalPhysiology, 37, 911-917) from lipoproteins or tissues in the presence ofBHT. The combined extracts were rapidly dried under nitrogen,resuspended in methanol:H₂O (98:2, v:v), and then neutral lipids in thelipid extracts were removed by passage through a 18C minicolumn(Supelclean LC-18 SPE tubes, 3 ml; Supelco Inc., Bellefonte, Pa.). Aknown amount of dimyristyl phosphatidyl choline (DMPC) was added to thepolar lipid fraction as an internal standard, and the lipids were driedunder nitrogen and stored under an argon atmosphere at −80° C. untilanalysis within 24 h. Calibration curves were constructed with a fixedamount of DMPC and varying mol % of each synthetic oxidized PC speciesand used to correct for the differences in ionization response factorsobserved amongst the different lipids. In additional preliminary studiesthe quantification methods employed were independently validated foreach analyte by demonstrating identical results to those obtained by themethod of standard additions

Results

Quantification of various specific oxidated PC species by LC/ESI/MS/MSanalysis in native and oxidized forms of LDL revealed substantialincreases in the content of oxidated phosphatidyl choline species (FIG.7a , data for native LDL, NO₂-LDL shown). Regardless of what time pointof oxidation was examined, HODA-PC and HOOA-PC were major products ofLDL oxidation by MPO. The combined mol % (relative to remainingunoxidized phospholipids) and ND-PC) detected in NO₂-LDL (FIG. 7a )correspond to ˜1.2 mol %. Of these, the combined content of the 8oxidated PC species quantified in NO₂LDL preparation (FIG. 7a )correspond to 0.73 mol %.

To determine if oxidated PC species are formed in vivo, thoracic aortaewith and without extensive atherosclerotic lesions were isolated fromWatanabe heritable hyperlipidemic (WHHL) rabbits and the levels ofmultiple distinct specific oxidized phospholipids were determined usingLC/ESI/MS/MS analyses. Significant increases in the content of each ofthe oxidated PCs derived from oxPAPC (HOOA-PC, KOOA-PC, HOdiA-PC,KOdiA-PC) and oxPLPC (HODA-PC, KODA-PC, HDdiA-PC and KDdiA-PC) werenoted in the diseased vessels (FIG. 7b ). Interestingly, while thelevels of oxidated PC species derived from PLPC were lower than thatobserved for the more highly oxidized ON-PC and ND-PC, levels ofoxidated PC species derived from PAPC were comparable to that observedfor OV-PC and G-PC (FIG. 7a ).

Presence of HETEs, HODEs, F2 Isoprostanes and Oxidated PC Species inAtherscloerotic Lesions of Human Subjects

The Angiogard is an emboli-protection device recently invented for useduring percutaneous vascular interventions. It is deployed distal to thetarget lesion prior to balloon inflation for angioplasty. It serves as atemporary umbrella, catching extruded lipid-rich plaque material throughan inert sieve-like mesh. The pores of the mesh are large and microscopyconfirms that they do not obstruct flow of blood cells or platelets, butrather capture large lipid globules. The material captured in theAngiogard at the time of intervention was analyzed to determine thelipid species in the plaque material. FIG. 8 shows the levels ofmultiple distinct lipid oxidation products quantified by LC/ESI/MS/MSmethods in plaque material recovered from the Angiogard. For comparison,we also assessed the levels of the same oxidized lipids in normal aorticintima recovered at the time of organ harvest from heart transplantdonors. Dramatic increases in F₂-Isoprostanes and each of the HETEsmonitored were observed. Analysis of plaque material captured in theAngiogard also confirmed detection of multiple distinct oxPC species(data not shown).

Methods of Determining Levels of Select Myeloperoxidase-GeneratedOxidation Products A. Dityrosine and Nitrotyrosine

Dityrosine and nitrotyrosine levels in the bodily sample can bedetermined using monoclonal antibodies that are reactive with suchtyrosine species. For example, anti-nitrotyrosine antibodies may be madeand labeled using standard procedures and then employed in immunoassaysto detect the presence of free or peptide-bound nitrotyrosine in thesample. Suitable immunoassays include, by way of example,radioimmunoassays, both solid and liquid phase, fluorescence-linkedassays or enzyme-linked immunosorbent assays. Preferably, theimmunoassays are also used to quantify the amount of the tyrosinespecies that is present in the sample.

Monoclonal antibodies raised against the dityrosine and nitrotyrosinespecies are produced according to established procedures. Generally, thedityrosine or nitrotyrosine residue, which is known as a hapten, isfirst conjugated to a carrier protein and used to immunize a hostanimal. Preferably, the dityrosine and nitrotyrosine residue is insertedinto synthetic peptides with different surrounding sequence and thencoupled to carrier proteins. By rotating the sequence surrounding thedityrosine and nitrotyrosine species within the peptide coupled to thecarrier, antibodies to only the dityrosine and nitrotyrosine species,regardless of the surrounding sequence context, are generated. Similarstrategies have been successfully employed with a variety of other lowmolecular weight amino acid analogues.

Suitable host animals, include, but are not limited to, rabbits, mice,rats, goats, and guinea pigs. Various adjuvants may be used to increasethe immunological response in the host animal. The adjuvant useddepends, at least in part, on the host species. To increase thelikelihood that monoclonal antibodies specific to the dityrosine andnitrotyrosine are produced, the peptide containing the respectivedityrosine and nitrotyrosine species may be conjugated to a carrierprotein which is present in the animal immunized. For example, guineapig albumin is commonly used as a carrier for immunizations in guineapigs. Such animals produce heterogenous populations of antibodymolecules, which are referred to as polyclonal antibodies and which maybe derived from the sera of the immunized animals.

Monoclonal antibodies, which are homogenous populations of an antibodythat binds to a particular antigen, are obtained from continuous cellslines. Conventional techniques for producing monoclonal antibodies arethe hybridoma technique of Kohler and Millstein (Nature 356:495-497(1975)) and the human B-cell hybridoma technique of Kosbor et al(Immunology Today 4:72 (1983)). Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, Iga, IgD and any classthereof. Procedures for preparing antibodies against modified aminoacids, such as for example, 3-nitrotyrosine are described in Ye, Y. Z.,M. Strong, Z. Q. Huang, and J. S. Beckman. 1996. Antibodies thatrecognize nitrotyrosine. Methods Enzymol. 269:201-209.

In general, techniques for direct measurement of protein bounddityrosine and nitrotyrosine species from bodily fluids involves removalof protein and lipids to provide a fluid extract containing free aminoacid residues. The tissues and bodily fluids are stored, preferably inbuffered, chelated and antioxidant-protected solutions, preferably at−80° C. as described above. The frozen tissue, and bodily fluids arethen thawed, homogenized and extracted, preferably with a single phasemixture of methanol:diethylether:water as described above to removelipids and salts. Heavy isotope labeled internal standards are added tothe pellet, which, preferably, is dried under vacuum, hydrolyzed, andthen the amino acid hydrolysate resuspended, preferably in awater:methanol mixture, passed over a mini solid-phase C18 extractioncolumn, derivatized and analyzed by stable isotope dilution gaschromatography-mass spectrometry as above. Values of free dityrosine andnitrotyrosine species in the bodily sample can be normalized to proteincontent, or an amino acid such as tyrosine as described above.

In a highly preferred procedure, protein is delipidated and desaltedusing two sequential extractions with a single phase mixture ofH₂O/methanol/H₂O-saturated diethyl ether (1:3:8 v/v/v). Oxidizedtyrosine standards (2 μmol each) and universal labeled tyrosine (2 nmol)are added to protein pellets. Proteins are hydrolyzed by incubating thedesalted protein pellet with degassed 6N HCl supplemented with 1% phenolfor 24 h under argon atmosphere. Amino acid hydrolysates are resuspendedin chelex treated water and applied to mini solid-phase C18 extractioncolumns (Supelclean LC-C18SPE minicolumn; 3 ml; Supelco, Inc.,Bellefonte, Pa.) pre-equilibrated with 0.1% trifluoroacetic acid.Following sequential washes with 2 ml of 0.1% trifluoroacetic acid,oxidized tyrosines and tyrosine are eluted with 2 ml 30% methanol in0.1% trifluoroacetic acid, dried under vacuum and then analyzed by massspectrometry.

Tandem mass spectrometry is performed using electrospray ionization anddetection with an ion trap mass spectrometer (LCQ Deca, ThermoFinigann,San Jose, Calif.) interfaced with a Thermo SP4000 high performanceliquid chromatograph (HPLC). Samples are suspended in equilibrationsolvent (H₂O with 0.1% formic acid) and injected onto a Ultrasphere C18column (Phenominex, 5 μm, 2.0 mm×150 mm). L-Tyrosine and its oxidationproducts are eluted at a flow rate of 200 μl/min using a linear gradientgenerated against 0.1% formic acid in methanol, pH 2.5 as the secondmobile phase. Analytes are monitored in positive ion mode with full scanproduct ion MS/MS at unit resolution. Response is optimized with a sprayvoltage setting of 5 KV and a spray current of 80 μA. The heatedcapillary voltage is set at 10 V and the temperature to 350° C. Nitrogenis used both as sheath and auxillary gas, at a flow rate of 70 and 30arbitrary units, respectively. The analyte abundance is evaluated bymeasuring the chromatographic peak areas of selected product ionsextracted from the full scan total ion chromatograms, according to thecorresponding ion trap product ion spectra. The ions monitored for eachanalyte are: 3-nitro[¹²C₆]tyrosine (mass-to-charge-ratio (m/z) 227, 181and 210), 3-nitro[¹³C₆]tyrosine (m/z 233, 187 and 216), 3-nitro[¹³C₉¹⁵N₁]tyrosine (m/z 237, 190 and 219), [¹²C₆]tyrosine (m/z 182, 136 and165), [¹³C₉ ¹⁵N₁]tyrosine (m/z 192, 145 and 174). Tyrosine andnitrotyrosine are base-line resolved under the HPLC conditions employed,permitting programming of the LCQ Deca for analysis over 0-7 min fordetection of tyrosine isotopomers, and from 7 min on for detection of3-nitrotyrosine isotopomers.

Free nitrotyorsine and dityrosine are similarly measured in samples, buttissue or bodily fluid is first passed through a low molecular weightcut off filter and the low molecular weight components analyzed byLC/ECS/MS/MS. Values of free and protein-bound dityrsoine andnitrotyrosine species in the bodily sample can be normalized to proteincontent, or an amicon acid such as the precursor tyrosine, as describedbelow.

B. Lipid Oxidation Products

Lipid oxidation products can be measured by HPLC with UV detection orHPLC with on line mass spectrometry. Other analytical methods includingGC/MS and immunocytochemical methods may also be used. F2Isoprostanesare measurable by various mass spectrometry techniques as known in theart.

Methods of extracting and quantifying the MPO-generated lipid oxidationproducts hydroxy-eicosatetraenoic acids (HETEs), hydroxy-octadecadienoicacids (HODEs), F2Isoprostanes; the 5-oxovaleric acid esters of 2-lysoPC(OV-PC); 5-cholesten-5α, 6α-epoxy-3β-ol (cholesterol α-epoxide);5-cholesten-5β, 6β-epoxy-3β-ol (cholesterol β-epoxide);5-cholesten-3β,7β-diol (7-OH-cholesterol); 5-cholesten-3β, 25-diol(25-OH cholesterol 5-cholesten-3β-ol-7β-hydroperoxide (7-OOHcholesterol); and cholestan-3β, 5α, 6β-triol (triol). are described inSchmitt, et al. (1999) Biochemistry, Vol. 38, 16904-16915, which isspecifically incorporated herein by reference. For determination of9-H(P)ODE, 9-H(P)ETE and F₂-isoprostanes, hydroperoxides in reactionmixtures are reduced to their corresponding hydroxides during extractionutilizing a modified Dole procedure in which the reducing agent,triphenylphosphine, is present (Savenkova, M. L., et al. (1994) J. Biol.Chem. 269, 20394-20400). These conditions also inhibit artifactualformation of isoprostanes and oxidized lipids. Lipids are dried underN₂, resuspended in isopropanol (2 ml) and then fatty acids released bybase hydrolysis with 1 N sodium hydroxide (2 ml) at room temperatureunder N₂ for 90 min. The samples are acidified (pH 3.0) with 2N HCl,known amounts of internal standards are added and free fatty acids areextracted twice with hexane (5 ml). The content of 9-H(P)ODEs,9-H(P)ETEs and F₂-isoprostanes are then determined by LC/MS/MS analysisas outlined below.

1-palmitoyl-2 oxovaleryl-sn-glycero-3-phosphatidyl choline (PoxvPC) isextracted by the same modified Dole procedure used for 9-H(P)ODE,9-H(P)ETE and F₂ isoprostane analyses as above, but omitting addition ofthe reductant, triphenylphosphine. Lipids are dried under N₂,resuspended in methanol and stored under argon at −70° C. untilsubsequent LC/MS analysis as outline below. Sterol oxidation productsare extracted by adding 4 M NaCl (150 μl) and acetonitrile (500 μl).Samples are vortexed, centrifuged, and the upper organic phase removed.Extracts are dried under N₂, resuspended in methanol, and stored underargon at −70° C. until analysis by HPLC with on-line mass spectrometricanalysis.

Mass spectrometric analyses are performed on a Quatro II triplequadruple mass spectrometer interfaced with an HP 1100 HPLC.F₂-isoprostanes are quantified by stable isotope dilution massspectrometry using on-line reverse phase HPLC tandem mass spectrometry(LC/MS/MS) with 8-epi-[²H₄]PGF_(2α) as standard as described by Mallat(Mallat, Z., et al. (1999) J. Clin. Invest. 103, 421-427). For 9-HODEand 9-HETE analyses, lipid extracts generated following base hydrolysisof reduced lipids (above) are dried under N₂ and reconstituted inmethanol. An aliquot of the mixture is then injected on an UltrasphereODS C18 column equilibrated and run under isocratic conditions employingmethanol:H₂O, (85:15, v/v) as solvent. Column eluent is split (930μl/min to UV detector and 70 μl/min to mass detector) and analyzed bythe mass spectrometer. LC/MS/MS analysis of 9-HODE, 9-HETE andF₂-isoprostanes in column effluents is performed using electrosprayionization mass spectrometry (ESI-MS) in the negative-ion mode withmultiple reaction monitoring (MRM) and monitoring the transitions m/z295→171 for 9-HODE; m/z 319→151 for 9-HETE; m/z 353→309 forF₂-isoprostanes; and m/z 357→313 for [²H₄]PGF_(2α).

Quantification of POxvPC is performed on lipid extracts utilizing HPLCwith on-line ESI-MS analysis in the positive ion mode and selected ionmonitoring at m/z 782 and m/z 594, respectively. An aliquot of lipidextract reconstituted in methanol (above) is mixed 0.1% formic acid inmethanol (mobile phase B) and loaded onto a Columbus C18 column (1×250mm, 5 μm, P. J. Cobert, St. Louis, Mo.) pre-equilibrated in 70% mobilephase B, 30% mobile phase A (0.1% formic acid in water) at a flow rateof 30 μl/min. Following a 3 min wash period at 70% mobile phase B, thecolumn is developed with a linear gradient to 100% mobile phase B,followed by isocratic elution with 100% mobile phase B. Externalcalibration curves constructed with authentic POxvPC are used forquantification. 7-OH cholesterol, 7-keto cholesterol, and 7-OOHcholesterol are resolved on an Ultrasphere ODS C18 column. The elutiongradient consisted of 91:9, acetonitrile:water+0.1% formate (v:v), andthe column washed between runs with acetonitrile+0.1% formate. Columneffluent is split (900 μl/min to UV detector and 100 μl/min to massdetector) and ionized by atmospheric pressure chemical ionization (APCI)in the positive-ion mode with selected ion monitoring. Identification of7-OH cholesterol is performed by demonstrating co-migration of ions withm/z 385.3 (M−H₂O)⁺ and m/z 367.3 (M−2H₂O)⁺ with the same retention timeas authentic standard. The integrated area of the ion current for thepeak monitored at m/z 367.3 is used for quantification. Identificationof 7-OOH cholesterol is performed by demonstrating co-migration of ionswith m/z 401.3 (M−H₂O)⁺, m/z 383.3 (M−2H₂O)⁺ and m/z 367.3 (M−H₂O₂)⁺with the same retention time as authentic standard. The integrated areaof the ion current for the peak monitored at m/z 401.3 is used forquantification. Identification of 7-keto cholesterol is performed bydemonstrating co-migration of ions with m/z 401.3 (M+H)⁺ and m/z 383.3(M−H₂O)⁺ with the same retention time as authentic standard. Theintegrated area of the ion current for the peak monitored at m/z 401.3is used for quantification. External calibration curves constructed withauthentic 7-OH cholesterol, 7-OOH cholesterol and 7-keto cholesterol areused for quantification following preliminary APCI LC/MS experimentsdemonstrating identical results to those obtained by the method ofstandard additions. The retention times for 25-OH cholesterol, 5,6 α-and β-epoxides, and triol are determined by LC/MS analysis of authenticstandards.

Predetermined Value

The level of MPO mass, MPO activity, or select MPO-generated oxidationproduct in the bodily sample obtained from the test subject is comparedto a predetermined value. The predetermined value is based upon thelevels of MPO activity, MPO mass, or select MPO-generated oxidationproduct in comparable samples obtained from the general population orfrom a select population of human subjects. For example, the selectpopulation may be comprised of apparently healthy subjects. “Apparentlyhealthy”, as used herein, means individuals who have not previously hadany signs or symptoms indicating the presence of atherosclerosis, suchas angina pectoris, history of an acute adverse cardiovascular eventsuch as a myocardial infarction or stroke, evidence of atherosclerosisby diagnostic imaging methods including, but not limited to coronaryangiography. Apparently healthy individuals also do not otherwiseexhibit symptoms of disease. In other words, such individuals, ifexamined by a medical professional, would be characterized as healthyand free of symptoms of disease.

The predetermined value is related to the value used to characterize thelevel of MPO activity or MPO mass in the bodily sample obtained from thetest subject. Thus, if the level of MPO activity is an absolute valuesuch as the units of MPO activity per leukocyte or per ml of blood, thepredetermined value is also based upon the units of MPO activity perleukocyte or per ml of blood in individuals in the general population ora select population of human subjects. Similarly, if the level of MPOactivity or MPO mass is a representative value such as an arbitrary unitobtained from a cytogram, the predetermined value is also based on therepresentative value.

The predetermined value can take a variety of forms. The predeterminedvalue can be a single cut-off value, such as a median or mean. Thepredetermined value can be established based upon comparative groupssuch as where the risk in one defined group is double the risk inanother defined group. The predetermined can be a range, for example,where the general population is divided equally (or unequally) intogroups, such as a low risk group, a medium risk group and a high-riskgroup, or into quadrants, the lowest quadrant being individuals with thelowest risk the highest quadrant being individuals with the highestrisk.

The predetermined value can be derived by determining the level of MPOactivity or mass in the general population. Alternatively, thepredetermined value can be derived by determining the level of MPOactivity or mass in a select population, such as an apparently healthynonsmoker population. For example, an apparently healthy, nonsmokerpopulation may have a different normal range of MPO activity or MPO massthan will a smoking population or a population whose member have had aprior cardiovascular disorder. Accordingly, the predetermined valuesselected may take into account the category in which an individualfalls. Appropriate ranges and categories can be selected with no morethan routine experimentation by those of ordinary skill in the art.

Predetermined values of MPO activity or MPO mass, such as for example,mean levels, median levels, or “cut-off” levels, are established byassaying a large sample of individuals in the general population or theselect population and using a statistical model such as the predictivevalue method for selecting a positivity criterion or receiver operatorcharacteristic curve that defines optimum specificity (highest truenegative rate) and sensitivity (highest true positive rate) as describedin Knapp, R. G., and Miller, M. C. (1992). Clinical Epidemiology andBiostatistics. William and Wilkins, Harual Publishing Co. Malvern, Pa.,which is specifically incorporated herein by reference. A “cutoff” valuecan be determined for each risk predictor that is assayed. Thestandardized method that was used in Example 1 below employs theguaiacol oxidation assay as described in Klebanoff, S. J., Waltersdorph,A. N. and Rosen, H. 1984. “Antimicrobial activity of myeloperoxidase”.Methods in Enzymology. 105: 399-403).

Comparison of MPO Activity and Mass Levels and Levels of SelectMPO-Generated Oxidation Products in the Bodily Sample from the TestSubject to the Predetermined Value.

The levels of each risk predictor, i.e., MPO activity, MPO mass andselect MPO-generated oxidation product, in the individual's bodilysample may be compared to a single predetermined value or to a range ofpredetermined values. If the level of the present risk predictor in thetest subject's bodily sample is greater than the predetermined value orrange of predetermined values, the test subject is at greater risk ofdeveloping or having CVD than individuals with levels comparable to orbelow the predetermined value or predetermined range of values. Incontrast, if the level of the present risk predictor in the testsubject's bodily sample is below the predetermined value or range ofpredetermined range, the test subject is at a lower risk of developingor having CVD individuals with levels comparable to or above thepredetermined value or range of predetermined values. For example, atest subject who has a higher number of neutrophils or monocytes or bothwith elevated levels of MPO activity or MPO mass as compared to thepredetermined value is at high risk of developing cardiovasculardisease, and a test subject who has a lower number of neutrophils ormonocytes or both with decreased or lower levels of MPO activity or MPOmass as compared to the predetermined value is at low risk of developingcardiovascular disease. The extent of the difference between the testsubject's risk predictor levels and predetermined value is also usefulfor characterizing the extent of the risk and thereby, determining whichindividuals would most greatly benefit from certain aggressivetherapies. In those cases, wherein the predetermined value ranges aredivided into a plurality of groups, such as the predetermined valueranges for individuals at high risk, average risk, and low risk, thecomparison involves determining into which group the test subject'slevel of the relevant risk predictor falls.

The present diagnostic tests are useful for determining if and whentherapeutic agents which are targeted at preventing CVD should andshould not be prescribed for a patient. For example, individuals withvalues of MPO activity (U/mg PMN protein; or U/ml blood) above a certaincutoff value, or that are in the higher tertile or quartile of a “normalrange,” could be identified as those in need of more aggressiveintervention with lipid lowering agents, life style changes, etc.

One of the most attractive findings of increased MPO as a predictor ofrisk for CVD is that it represents an independent marker to identifyindividuals with increased risk for cardiovascular disease. That is, inmultivariate analyses vs. other known risk factors for CVD (e.g. lipidlevels such as LDL, HDL, total cholesterol, triglycerides, as well asfamily history, tobacco use, hypertension, diabetes), elevated levels ofMPO activity and mass independently predicted association with CVD.Thus, the present diagnostic tests are especially useful to identifyindividuals at increased risk who might otherwise not have beenidentified by existing screening protocols/methods. Moreover, thepresent risk predictors can be used in combination with currently usedrisk predictors, such as blood LDL levels, blood triglyceride levels andblood C-reactive protein levels, and algorithms based thereon to moreaccurately characterize an individual's risk of developing or havingCVD.

Evaluation of CVD Therapeutic Agents

The present diagnostic tests are also useful for evaluating the effectof CVD therapeutic agents on patients who have been diagnosed as havingor as being at risk of developing CVD. Such therapeutic agents include,but are not limited to, anti-inflammatory agents, insulin sensitizingagents, antihypertensive agents, anti-thrombotic agents, anti-plateletagents, fibrinolytic agents, lipid reducing agents, direct thrombininhibitors, ACAT inhibitor, CDTP inhibitor thioglytizone, andglycoprotein II b/IIIa receptor inhibitors. Such evaluation comprisesdetermining the levels of one or more of the present risk predictorsincluding MPO activity, MPO mass, select MPO-generated oxidationproducts, and combinations thereof, in a bodily sample taken from thesubject prior to administration of the therapeutic agent and acorresponding bodily fluid taken from the subject followingadministration of the therapeutic agent. A decrease in the level of theselected risk factor in the sample taken after administration of thetherapeutic as compared to the level of the selected risk factor in thesample taken before administration of the therapeutic agent isindicative of a positive effect of the therapeutic agent oncardiovascular disease in the treated subject.

EXAMPLES

The following examples are for purposes of illustration only and are notintended to limit the scope of the claims which are appended hereto.

Example 1 Levels of MPO Activity and MPO Mass in Blood Samples ofPatients with and without Coronary Artery Disease Methods StudyPopulation:

Based on logistic regression power calculations (assuming equal sizegroups), 326 patients were needed to provide 80% power (α=0.05) todetect a statistically significant odds ratio of at least 2.0 for highMPO (upper quartile). Subjects (n=333) were identified from twopractices within the Cardiology Department of the Cleveland ClinicFoundation. First, a series of 85 consecutive patients were enrolledfrom the Preventive Cardiology Clinic. Simultaneously, 125 consecutivepatients were enrolled from the catheterization laboratory. Based uponCAD prevalence in this series, a need for 116 additional controlsubjects was determined. All patients who did not have significant CADupon catheterization over the preceding 6 months were identified fromthe catheterization database, and then 140 were randomly selected (basedupon area code/telephone number) and invited to participate for MPOmeasurement. CAD was defined by a history of documented myocardialinfarction, prior coronary revascularization intervention (CABG orpercutaneous coronary intervention), or as the presence of ≧50% stenosisin one or more coronary arteries identified during cardiaccatheterization. Exclusion criteria for the CAD group were an acutecoronary event within 3 months preceding enrolment, end stage renaldisease and bone marrow transplantation. The control group consisted ofsubjects who had undergone diagnostic coronary angiography that revealedno evidence of significant CAD. Exclusion criteria for control subjectswere one or more coronary vessels with stenosis ≧50%, valvular heartdisease, left ventricle dysfunction, end-stage renal disease, bonemarrow transplantation, or evidence of infection or active inflammatorydiseases as revealed by history and exam. All patients were older than45 years of age and afebrile. Clinical history was assessed for diabetesmellitus, smoking history past and present, hypertension and whether anyfirst-degree relatives had CAD (men by the age of 50 years and femalesby the age of 60). Study protocol and consent forms were approved by theCleveland Clinic Foundation Institutional Review Board and informed,written consent was obtained from all subjects. Samples were coded toensure anonymity and all analyses were performed in a blinded fashion.

Measurements:

Blood was drawn following an overnight fast into EDTA-containing tubesand used to quantify WBC, low density lipoprotein cholesterol (LDLc),high density lipoprotein cholesterol (HDLc), total cholesterol (TC) andfasting triglycerides (TG). Neutrophils were isolated by buoyant densitycentrifugation (Hazen, S. L., et al., J. Biol. Chem. 271:1861-1867).Cell preparations were at least 98% homogeneous by visual inspection.Leukocyte preparations were supplemented to 0.2% cetyltrimethylammoniumbromide for cellular lysis, incubated at room temperature for 10 min,snap frozen in liquid nitrogen and stored at −80° C. until analysis.

Functional MPO was quantified by peroxidase activity assay of neutrophillysates. Briefly, detergent-lysed cells (10⁴/ml; triplicate samples)were added to 20 mM phosphate buffer (pH 7.0) containing 14.4 mMguaiacol, 0.34 mM H₂O₂, and 200 μM DTPA and the formation of guaiacoloxidation product monitored at A₄₇₀ at 25° C. (Klebanoff, S. J., et al.,Methods Enzymol. 105:399-403, Capeillere-Blandin, C., Biochem. J.36(Pt2):395-404).. A millimolar absorbance coefficient of 26.6 mM⁻¹ cm⁻¹for the diguaiacol oxidation product was used to calculate peroxidaseactivity where one unit of MPO activity is defined as the amount thatconsumes 1 μmol of H₂O₂ per minute at 25° C. MPO activity reported isnormalized either per mg of neutrophil protein (Leukocyte-MPO) or per mlof blood (Blood-MPO). Blood-MPO (Units MPO per ml of blood) wasestimated by multiplying the units of MPO activity per neutrophil timesthe absolute neutrophil count (per microliter blood) times 1000. Proteinconcentration was determined as described (Markwell, M. A., et al.,Anal. Biochem. 87:206-210).

Levels of Leukocyte-MPO in an individual were found to be extremelyreproducible, demonstrating less than ±7% variations in subjects overtime (n=6 males evaluated once per 1-3 months for ≧2 year period). Thecoefficient of variance for determination of Leukocyte-MPO, asdetermined by analysis of samples multiple times consecutively, was4.2%. Leukocyte-MPO determination for 10 samples run on 3 separate daysyielded a coefficient of variance of 4.6%. The coefficient of variancefor determination of Blood-MPO as determined by analysis of samplesmultiple times consecutively, was 4.2%. Blood-MPO determination for 10samples run on 3 separate days yielded a coefficient of variance of4.8%. MPO mass per neutrophil was determined using an enzyme linkedimmunosorbent assay (ELISA). Capture plates were made by incubating96-well plates overnight with polyclonal antibody (Dako, Glostrup,Denmark.) raised against the heavy chain of human MPO (10 μg/ml in 10 mMPBS, pH 7.2). Plates were washed and sandwich ELISA performed onleukocyte lysates using alkaline phosphatase-labeled antibody to humanMPO. MPO mass was calculated based on standard curves generated withknown amounts of human MPO purified from leukocytes as described (Hazen,S. L., et al., J. Biol. Chem. 271:1861-1867). Purity of isolated MPO wasestablished by demonstrating a RZ of 0.87 (A₄₃₀/A₂₈₀), SDS PAGEanalysis, and in-gel tetramethylbenzidine peroxidase staining (Podrez,E. A., et al., J. Clin. Invest 103:1547-1560). Enzyme concentration wasdetermined spectrophotometrically utilizing an extinction coefficient of89,000 M⁻¹ cm⁻¹/heme.

Statistical Analysis:

Presentation characteristics are depicted as either mean±standarddeviation or median (interquartile range) for continuous measures andnumber and percent for categorical measures. Differences between CAD andcontrol subjects were evaluated with Wilcoxon rank sum or chi-squaretests. MPO levels were divided into quartiles for analyses becauseneither Leukocyte-MPO nor Blood-MPO activity follows a Gaussiandistribution. Unadjusted trends for increasing CAD rates with increasingMPO activity were evaluated with the Cochran-Armitage trend test. Amodified Framingham Global Risk score was determined utilizing adocumented history of hypertension rather than the recorded bloodpressure at time of catheterization (Taylor, A. J., et al., Circulation101:1243-1248).

Logistic regression models (SAS System, SAS Institute, Cary N.C.) weredeveloped to calculate odds ratios (OR) estimating the relative riskassociated with the combined 2^(nd) and 3^(rd) quartiles of MPO activityand the highest quartile of MPO activity compared to the lowestquartile. Adjustments were made for individual traditional CAD riskfactors (age, gender, diabetes, hypertension, smoking (ever or current),family history, TC, LDLc, HDLc, TG, WBC). Hosmer-Lemeshow goodness offit tests were employed to evaluate appropriate model fit. Associationsamong continuous variables were assessed with use of Spearman'srank-correlation coefficient. Associations among categorical variableswere assessed using Wilcoxon rank sum tests.

Results Patient Demographics:

The clinical and biochemical characteristics of subjects thatparticipated in this study are shown in Table 1. Subjects with CAD wereolder, more likely to be male, and more likely to have a history ofdiabetes, hypertension and smoking. CAD subjects also exhibitedincreased fasting triglyceride levels, increased use of lipid loweringmedications (predominantly statins), aspirin and other cardiovascularmedications. Consistent with other studies, Framingham Global RiskScore, absolute neutrophil count and WBC were significantly increased insubjects with CAD (p<0.001 for each; Table 1).

TABLE 1 Clinical and Biochemical Characteristics of Subjects c) Controli) CAD Characteristics (n = 175) (n = 158) Age, y  55 ± 10  64 ± 13***Gender (female), % 42 20*** Diabetes^(†), %  5 23*** Hypertension^(‡), %31 58*** Family history of CAD, % 53 54 History of smoking, % 49 78***Current smoking^(Φ), % 10  9 Any lipid lowering medications, % 27 70***Statin, % 25 65*** ASA, % 71 84** ACE Inhibitors, % 18 44*** BetaBlockers, % 27 59*** Calcium Channel Blockers, % 15 24* Totalcholesterol, mg/dL^(¶) 203 (166-234) 203 (174-234) LDL cholesterol,mg/dL^(¶) 132 (89-144) 122 (90-146) HDL cholesterol, mg/dL^(¶)  49(40-56)  43 (36-49) Fasting triglycerides, mg/dL^(§) 121 (91-198) 159(117-240)*** WBC (×10³/mm³) 7.4 ± 3.0 8.4 ± 3.2*** ANC (×10³/mm³) 3.8 ±1.9 5.2 ± 2.6*** Framingham Global Risk 5.5 ± 3.8 8.0 ± 3.0***Stratification of Leukocyte-MPO, Blood-MPO and White Blood Cell CountVs. Prevalence of Coronary Artery Disease:

To test the hypothesis that individuals with higher levels of MPO have ahigher prevalence of CAD, we isolated neutrophils and measured their MPOcontent. MPO activity per mg of neutrophil protein (Leukocyte-MPO)differed significantly by CAD status with a median of 13.4 U/mg forcontrol subjects vs. 18.1 U/mg for CAD patients (p<0.001 for trend, andfor difference; FIG. 1). Stratification of Leukocyte-MPO levels byquartiles for the entire cohort revealed a positive correlation with CADstatus (p<0.001 for trend) with individuals in the highest quartilehaving the highest risk (OR(CI), 8.8 (4.4-17.5); Table 2). In additionto quantifying leukocyte MPO content by its catalytic activity (i.e. afunctional assay), we independently quantified MPO mass per neutrophilin a random subset of subjects (n=111) using an enzyme linkedimmunosorbent assay. Results observed from this assay significantlycorrelated (r=0.95) with the activity measurements (data not shown).Since rates for CAD in the second and third quartiles of Leukocyte-MPOappeared comparable (Table 2), they were combined for all furtheranalyses and are referred to as the mid range levels in univariate andmultivariate models. As has been seen in other studies, FraminghamGlobal Risk Score and WBC were likewise positively correlated with ratesof CAD (Table 2).

TABLE 2 Odds Ratio of Coronary Artery Disease Prevalence According toMyeloperoxidase Levels, White Blood Cell Count and Framingham GlobalRisk Score 1.      2) Quartile      a. 1 2 3 4 for trendLeukocyte-MPO^(†) U/mg PMN protein ≦11.8 11.9-15.3 15.4-19.8 ≧19.9 CADRate 24/91 (26%) 35/76 (46%) 36/83 (43%) 63/83 (76%) p < 0.001Unadjusted OR (CI) 1.0 2.4 (1.2-4.6)** 2.1 (1.1-4.0)* 8.8 (4.4-17.5)***2. Model 1^(a) OR (CI) 8.5 (3.7-19.7)*** 20.3 (7.9-52.1)*** 3. Model2^(b) OR (CI) 4.2 (2.1-8.1)*** 11.9 (5.5-25.5)*** ^(4.) Blood-MPO^(‡)U/PMN × ANC ≦2.9 3.0-4.1 4.2-5.7 ≧5.8 CAD Rate 16/91 (18%) 35/83 (42%)41/79 (52%) 66/80 (83%) p < 0.001 Unadjusted OR (CI) 1.0 3.4(1.7-6.8)*** 5.1 (2.5-10.2)*** 22.1 (10.0-48.7)*** i. Mo 3.6(1.8-7.5)*** 15.1 (6.2-36.7)*** ii. Model^(b) 5.3 (2.7-10.5)*** 20.4(8.9-47.2)*** iii. WB ×10³/mm³ ≦5.78 5.79-7.32 7.33-9.02 ≧9.03 CAD Rate24/85 (28%) 46/82 (56%) 38/83 (46%) 50/83 (60%) p < 0.001 Unadjusted OR(CI) 1.0 3.2 (1.7-6.2)*** 2.1 (1.1-4.1)* 3.9 (2.0-7.3)*** iv. Adjuste3.0 (1.6-5.7)*** 4.3 (2.1-8.9)*** v. Fra Global Risk Score ≦4 5-7 8-9≧10 CAD Rate 25/86 (29%) 41/114 (36%) 41/63 (65%) 51/70 (73%) p < 0.001Unadjusted OR (CI) 1.0 1.4 (0.8-2.5) 4.5 (2.3-9.1)*** 6.5 (3.2-13.2)***Adjusted^(c) OR (CI) 1.8 (1.0-3.3) 7.8 (3.5-17.5)***

The total content of MPO in blood is dependent on both MPO levels perleukocyte as well as the total number of leukocytes. Since neutrophilspossess >95% of the MPO content in blood, we estimated the level of MPOper ml of blood (Blood-MPO) by multiplying the content of MPO perneutrophil times the absolute neutrophil count. Rates of CAD werepositively correlated with Blood-MPO quartiles (p<0.001 for trend; FIG.9, Table 2).

Leukocyte-MPO is not Significantly Correlated with Traditional CoronaryArtery Risk Factors:

Possible correlations between traditional CAD risk factors andLeukocyte-MPO were next assessed. Leukocyte-MPO levels were independentof age, gender, diabetes, hypertension, smoking (ever or current), WBC,triglycerides LDLc and Framingham Global Risk. Weak negativecorrelations between Leukocyte-MPO and both total cholesterol (r=−0.15,p=0.005) and HDLc (r=−0.14, p−0.01) were observed. A positiveassociation was seen between Leukocyte-MPO and absolute neutrophil count(r=0.20, p<0.001) and family history of CAD (median leukocyte-MPO withfamily history=15.9 vs. 14.1 without, p=0.05). Similar correlations werenoted for Blood-MPO.

Leukocyte-MPO and Blood-MPO are Strongly Correlated with Coronary ArteryDisease Status Following Adjustments for Single and Multiple RiskFactors:

To evaluate whether Leukocyte-MPO and Blood-MPO independently associatewith CAD status, odds ratios for Leukocyte-MPO and Blood-MPO quartileswere adjusted for individual traditional CAD risk factors. Odds ratiosfor both the middle (2^(rd) plus 3^(rd)) and highest (4^(th)), relativeto the lowest (1^(st)), quartiles of both Leukocyte-MPO and Blood-MPOremained highly correlated with CAD status following adjustments forindividual traditional CAD risk factors, WBC and Framingham Global RiskScore (data not shown), with odds ratios ranged from 8.4 (CI=4.2-16.9,p<0.001) after adjustment for HDLc to 13.5 (CI=6.3-29.1, p<0.001) afteradjustment for smoking. Diabetes, hypertension, smoking, and to a lesserdegree age, HDLc, Framingham Global Risk and WBC, also remainedsignificant predictors for CAD status following single factoradjustments. Similar results were observed for Blood-MPO followingsingle factor adjustments for individual traditional CAD risk factors(data not shown).

Multivariable regression analyses were then performed using severalmodels (Table 2, FIG. 10). Model 1 examined Leukocyte- and Blood-MPOfollowing simultaneous adjustment for each of the single risk factorsthat were significantly correlated to CAD in the preceding step (i.e.,univariate regression). Leukocyte-MPO remained the strongest predictorof CAD status with an adjusted OR of 8.5 (CI=3.7-19.7, mid vs. lowquartile) and 20.3 (CI=7.9-52.1, high vs. low quartile). The adjustedodds ratio for WBC, a marker that predicts increased risk for CAD (2; 3;23-25), was 1.1 (CI=1.02-1.21). A second regression model adjusting forFramingham Global Risk Score and WBC yielded ORs for Leukocyte-MPO thatwere consistent with the large OR observed in Model 1 (mid vs. lowOR=4.2; high vs. low OR=11.9). The adjusted OR for Framingham GlobalRisk Score and WBC were also significant. Blood-MPO likewise remained astrong predictor of CAD status following multivariable adjustmentscompared to traditional CAD risk factors, Framingham Global Risk Scoreand WBC (Table 2)

Example 2 Flow Cytometric Analysis of Blood Samples from Subjects withand without CAD

Blood samples from patients whose leukocytes have above normal or belownormal levels of MPO were analyzed by flow cytometry. Whole blood fromeach patient was injected into a hematology analyzer that identifiesleukocytes based upon in situ cytochemical peroxidase staining (theAdvia 120 from Bayer). In the instrument, whole blood is first lysed andthe intact WBCs heated/fixed with formaldehyde. Peroxidase substrates(hydrogen peroxide and a chromophore) are then incubated with theleukocytes, and the resultant stained cells examined by flow cytometry(20 sec overall time between injection of sample and cytogram obtained).The results are shown in FIG. 11. The clusters of cells shown indifferent colors refer to: 1) Purple—neutrophils; 2) Green—monocytes; 3)Dark Blue—Lymphocytes; 4) Yellow—eosinophils; 5) Turquoise—largeunstained cells; 6) White—RBC Ghosts/noise. Based upon these data, thetotal white blood cell count (WBC) and a differential (% distribution ofneutrophils, monocytes, eosinophils and lymphocytes) are reported.

The location of a given cell cluster's position on the cytogram isrelated to its intensity of light absorption (Y axis—a property that isrelated to peroxidase activity, and hence, intensity of staining) andlight scatter (X axis—a property that is related to both size andgranularity/refractive index, properties linked to peroxidase activityand staining).

The left panel illustrates the cytogram from an individual whose MPOlevel per neutrophil (aka leukocyte-MPO) is below the average in apopulation (e.g. bottom 25%). The right panel illustrates the locationof the cytogram from an individual whose MPO level per neutrophil (akaleukocyte-MPO) is above average in a population (e.g. 50-75^(th)%). Notethat the location of the neutrophil cluster on the X and Y axes differ,and in general, higher MPO is shifted to the right. Also, the tilt ofthe major axis of the ellipse that comprises the neutrophil clusterdiffers. These changes carry information related to the content of MPOwithin that cell type.

Through use of modeling and standards with known peroxidase content, wecan develop standard curves to use this information to identify therelative level of peroxidase per leukocyte. The same kind of analysis ispossible for monocytes, the other major cell type in blood with MPO.Peroxidase staining in eosinophils is due to eosinophil peroxidase, arelated enzyme to MPO, but a different gene product.

Example 3 Dityrosine Levels in Blood from Human Subjects with andwithout CAD

The levels of protein-bound dityrosine were measured in blood samplesfrom 112 individuals with CAD and from 128 apparently healthy controlsubjects. The levels were measured by HPLC with on-line fluorescencedetection and were quantified using an external calibration curvegenerated with synthetic dityrosine. Results were normalized to thecontent of the precursor amino acid, tyrosine, which was simultaneouslyquantified by HPLC with on-line diode array detection. The resultsdemonstrated that subjects with CAD had higher levels (50% increased,P<0.001 for comparison of CAD vs. healthy subjects) of dityrosine intheir serum than that observed in serum from healthy age and sex-matchedsubjects.

Example 4 Nitrotyrosine Levels in Blood from Human Subjects with andwithout CAD

The levels of protein-bound 3-nitrotyrosine were measured in bloodsamples from the same subjects as Example 3 where 112 individuals withCAD and 128 apparently healthy control subjects were examined.Nitrotyrosine levels were measured by HPLC with on-line electrosprayionization tandem mass spectrometry (LC/ESI/MS/MS) using stable isotopedilution techniques. Results were normalized to the content of theprecursor amino acid, tyrosine, which was simultaneously quantified bystable isotope dilution LC/ESI/MS/MS. The results demonstrated thatsubjects with CAD had higher levels (2.8-fold increased, P<0.001 forcomparison of CAD vs. healthy subjects) of nitrotyrosine in their serumthan healthy age and sex-matched subjects.

Example 5 Blood Levels of HETEs, HODEs, and F2Isoprostanes in HumanSubjects with and without CAD

The levels of HETEs, HODEs and F2Isoprostanes were measured in bloodsamples from the same subjects as Example 3 where 112 individuals withCAD and 128 apparently healthy control subjects were examined. Lipidswere measured by HPLC with on-line electrospray ionization tandem massspectrometry (LC/ESI/MS/MS). Results were normalized to the content ofthe precursor lipid (arachidonic acid for HETEs and F2Isoprostanes, andlinoleic acid for HODEs), which were simultaneously quantified byLC/ESI/MS/MS. The results demonstrated that subjects with CAD had higherlevels of each of the oxidation products in their plasma than healthyage and sex-matched subjects. F2Isoprostane levels were 80% greater inplasma obtained from CAD vs. non-CAD subjects, P<0.001; levels of HETEsand HODEs were 60% greater in CAD vs. non-CAD subjects, P<0.001).

Example 6 Blood Levels of MPO-Generated Lipid Oxidation Products inHuman Subjects with and without CAD

The levels of phospholipid oxidation products shown to be generated byMPO (G-PC and ND-PC, the glutaric and nonanedioic monoesters of2-lysoPC; HDdiA-PC and HOdiA-PC, the 9-hydroxy-10-dodecenedioic acid and5-hydroxy-8-oxo-6-octenedioic acid esters of 2-lysoPC; HODA-PC andHOOA-PC, the 9-hydroxy-12-oxo-10-dodecenoic acid and5-hydroxy-8-oxo-6-octenoic acid esters of 2-lysoPC; KODA-PC and KOOA-PC,the 9-keto-12-oxo-10-dodecenoic acid and 5-keto-8-oxo-6-octenoic acidesters of 2-lysoPC; KDdiA-PC and KOdiA-PC, the 9-keto-10-dodecendioicacid and 5-keto-6-octendioic acid esters of 2-lysoPC; OV-PC and ON-PC,the 5-oxovaleric acid and 9-oxononanoic acid esters of 2-lysoPC; weremeasured in blood samples from 25 subjects with CAD and 12 apparentlyhealthy control subjects. In addition the levels of cholesterolα-epoxide, 5-cholesten-5α,6α-epoxy-3β-ol; cholesterol β-epoxide,5-cholesten-5β,6β-epoxy-3β-ol; 7-OH-cholesterol, 5-cholesten-3β,7β-diol;25-OH cholesterol, 5-cholesten-3β,25-diol; 7-OOH cholesterol,5-cholesten-3β-ol-7β-hydroperoxide; triol, cholestan-3β,5α,6β-triol.)were measured in blood samples from 25 subjects with CAD and 12apparently healthy control subjects. Lipids were measured by HPLC withon-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS)using established methods. Results were normalized to the content of theprecursor lipid (PAPC,1-hexadecanoyl-2-eicosatetra-5′,8′,11′,14′-enoyl-sn-glycero-3-phosphocholine;PLPC,1-hexadecanoyl-2-octadecadi-9′,12′-enoyl-sn-glycero-3-phosphocholine; orcholesterol), which were simultaneously quantified by LC/ESI/MS/MS. Theresults demonstrated that subjects with CAD had higher levels (50% to4-fold, depending upon the lipid) of each of the phospholipid oxidationproducts in their plasma than healthy age and sex-matched subjects.

1-22. (canceled)
 23. A system comprising: a) a plasma or serum samplefrom a subject having, or suspected of having, cardiovascular disease;b) reagents for detecting the level of myeloperoxidase in said plasma orserum sample, wherein said reagents comprise anti-myeloperoxidase (MPO)antibodies; and c) a table comprising predetermined values forcorrelating said level of MPO in said plasma or serum sample with therisk of cardiovascular disease (CVD), wherein at least a portion of saidpredetermined values define a plurality of CVD risk groups.
 24. Thesystem of claim 23, wherein said plasma or serum sample is a plasmasample.
 25. The system of claim 23, wherein said plasma or serum sampleis a serum sample.
 26. The system of claim 23, wherein said subject hascardiovascular disease.
 27. The system of claim 23, wherein said subjectis suspected of having cardiovascular disease.
 28. The system of claim23, wherein each of said CVD risk groups is defined by a range ofvalues.
 29. The system of claim 23, wherein said plurality of CVD riskgroups comprise a high risk group, a medium risk group, and a low riskgroup.
 30. The system of claim 23, wherein said plurality of CVD riskgroups comprise a low risk quadrant, second and third mid-rangequadrants, and a high risk quadrant.
 31. The system of claim 23, whereinsaid table further comprises white blood cell counts.
 32. The system ofclaim 23, wherein said predetermined values in said table were generatedfrom a population of patients with cardiovascular disease.